Molecular Sequence of Events and Signaling Pathways in Cerebral M

Here, we review the mechanisms by which primary cancer cells metastasize to the brain via a mechanism called epithelial-to-mesenchymal transition, as well as the involvement of certain m

NYMC Faculty Publications Faculty 4-1-2018

Molecular Sequence of Events and Signaling Pathways in Cerebral Metastases

New York Medical College

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Recommended Citation

Cooper, J B., Ronecker, J., Tobias, M., Mohan, A., Hillard, V., Murali, R., Gandhi, C., Schmidt, M., & Uniyal, M (2018) Molecular Sequence of Events and Signaling Pathways in Cerebral Metastases

Jhanwar-Anticancer Research, 38 (4), 1859-1877 https://doi.org/10.21873/anticanres.12424

This Article is brought to you for free and open access by the Faculty at Touro Scholar It has been accepted for inclusion in NYMC Faculty Publications by an authorized administrator of Touro Scholar For more information, please contact touro.scholar@touro.edu

D Gandhi, Meic H Schmidt, and Meena Jhanwar-Uniyal

This article is available at Touro Scholar: https://touroscholar.touro.edu/nymc_fac_pubs/1169

Abstract Brain metastases are the leading cause of

morbidity and mortality among cancer patients, and are

reported to occur in about 40% of cancer patients with

metastatic disease in the United States of America Primary

tumor cells appear to detach from the parent tumor site,

migrate, survive and pass through the blood brain barrier in

order to establish cerebral metastases This complex process

involves distinct molecular and genetic mechanisms that

mediate metastasis from these primary organs to the brain.

Furthermore, an interaction between the invading cells and

cerebral milieu is shown to promote this process as well.

Here, we review the mechanisms by which primary cancer

cells metastasize to the brain via a mechanism called

epithelial-to-mesenchymal transition, as well as the

involvement of certain microRNA and genetic aberrations

implicated in cerebral metastases from the lung, breast, skin,

kidney and colon While the mechanisms governing the

development of brain metastases remain a major hindrance

in treatment, understanding and identification of the

aforementioned molecular pathways may allow for improved

management and discovery of novel therapeutic targets

Brain metastases are a leading cause of morbidity and

mortality among cancer patients, and are reported to occur

in about 40% of cancer patients in the United States of

America (USA), with an incidence approaching 170,000/year

in the USA (1, 2) Primary organ tumors that have the

greatest propensity to metastasize to the brain include lung (50-60%), breast (15-20%), skin (5-10%), kidney (7%) and colon cancers (4-6%) (3, 4) In general, the median survival following a diagnosis of cerebral metastases is between 2 and 25 months, depending on the origin of the primary tumor and time of diagnosis (5, 6) The diversity of these primary sites suggests the possibility of a common mechanism by which these tumors metastasize to the brain Moreover, a complex interaction exists with the cerebral microenvironment that results in a propensity for these tumors to disseminate to the central nervous system (CNS) For many cancer patients, the diagnosis of metastasis to the brain can be devastating In some cases, only supportive care is recommended However, several studies show that there is a survival benefit to combined treatment of surgical resection and radiation therapy For example, a review of cerebral metastasis from gastroesophageal cancer showed a survival advantage in patients treated with resection of the metastatic lesion followed by radiation, which included whole-brain radiation therapy or stereotactic radiosurgery (7) They specifically reported a patient with a cerebellar metastasis diagnosed five months after treatment for his primary disease who had no recurrence five years after undergoing resection of the brain metastasis followed by

stereotactic radiosurgery Furthermore, Karagkiouzis et al.,

reported that in patients with solitary extrapulmonary metastasis from NSCLC who underwent surgical resection

of the primary tumor as well as the solitary metastasis had improved survival, especially if the metastasis was not present within less than six months from diagnosis (8) Despite current advances in treatment for metastatic lesions, including surgical resection, chemotherapy and radiation, there is limited benefit in the form of prolonged survival As such, improvement in therapeutic options for metastatic brain lesions remains an unmet necessity

Tumor cells bypass multiple checkpoints in order to establish metastasis to the brain A complex mechanism

This article is freely accessible online

Correspondence to: Dr Meena Jhanwar-Uniyal, 19 Skyline Drive,

Department of Neurosurgery, New York Medical College, Valhalla, NY

10595, U.S.A Tel: +1 9145942513, e-mail: meena_jhanwar@nymc.edu

Key Words: Brain metastases, epithelial-to-mesenchymal transition,

microRNA, genetic markers, signaling pathway, review

Review

Molecular Sequence of Events and Signaling

Pathways in Cerebral Metastases

JARED B COOPER, JENNIFER S RONECKER, MICHAEL E TOBIAS, AVINASH L MOHAN,

VIRANY HILLARD, RAJ MURALI, CHIRAG D GANDHI, MEIC H SCHMIDT and MEENA JHANWAR-UNIYAL

Department of Neurosurgery, WMCH/New York Medical College, Valhalla, NY, U.S.A.

termed epithelial-to-mesenchymal transition (EMT), which is

governed by a sequence of multiple signaling pathways,

appears to govern this process In addition, the presence of

cancer stem cells (CSC) within the tumor may contribute to

this process and aid in evading current chemotherapeutics.

Moreover, a distinctive genetic signature is associated with a

propensity for cerebral metastases unique to discrete primary

sites In this review, we discuss the molecular process and

genetic markers associated with the development of cerebral

metastases, with particular focus on the five primary tumor

sites, namely lung, breast, skin, colon and kidney, having the

most propensity for metastasizing to the brain.

Epithelial-to-mesenchymal Transition

in Cerebral Metastasis

In general, commencement of metastasis begins with

detachment of primary cancer cells from the tumor mass This

is followed by invasion through the basement membrane and

intravasation into the systemic hematologic and lymphatic

circulation The circulating tumor cells then extravasate

through gaps in endothelial cells at a distant site, where they

form a secondary lesion This complex sequence of metastasis

requires a sophisticated process, referred to as EMT The

process of EMT is well recognized as it plays an important

role in embryogenesis and later in severe wound healing EMT

is characterized by the loss of cellular apical-basal polarity,

resulting in the formation of a cell with mesenchymal

properties, allowing for dissociation of cell-cell interactions

and migratory potential Upon reaching the secondary site, the

cells appear to undergo a reversal process termed

mesenchymal-to-epithelial transition (MET) by which the

tumor cells regain phenotypic and genotypic properties of the

primary tissue This process is analogous in the development

of metastatic tumors Recent studies have shown that the

process of EMT can propel cancer cells into a CSC-like state

allowing them to acquire a mesenchymal phenotype,

suggesting a functional link between CSCs and the metastatic

process (9) Tam and Weinberg have attempted to elucidate a

detailed and complex transcriptional process that governs the

steps of EMT and its reversal mechanism MET (10) The

canonical EMT/MET processes are characterized by complex

genetic alteration that allows epithelial or mesenchymal cells

to be distinguished by expression of a number of classical

markers (11) Well-recognized epithelial markers include

cadherins and tight junction proteins such as E-cadherin.

Mesenchymal markers include the extracellular matrix

component fibronectin and the intermediate filament protein

vimentin During embryogenesis, EMT is governed by a

number of diverse growth factors including fibroblast growth

factor (FGF), platelet derived growth factor (PDGF),

epidermal growth factor (EGF) and transforming growth

factor beta (TGFβ), leading to activation of various receptor

tyrosine kinases (RTKs) (12) These mechanisms are shown

to reappear during the metastatic process via EMT, where

TGFβ plays a pivotal role.

Activation of the TGFβ pathway results in translocation of Smad transcription factor proteins into the nucleus where they interact with other transcription factors to activate or repress genes involved in EMT (13) TGFβ has a direct effect on EMT by down-regulation of epithelial and up-regulation of mesenchymal markers, through activation of a number of transcription factors including zinc finger SNAI1 (Snail), zinc finger SNAI2 (Slug), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box homeobox 2 (ZEB2), and twist family bHLH transcription factor 1 (TWIST), that are recognized as master regulators of this process (14) Under the influence of TGFβ, transcriptional repression of the transmembrane adhesion protein E-cadherin occurs, which emerged as a fundamental regulator in the process of tumor progression and EMT (12, 15, 16) Notably, transcription factors Snail, Slug and ZEB1/2 are recognized as key regulators in E-cadherin repression, in addition to their roles

in induction of mesenchymal genes (12, 16) GATA1, a known repressor of E-cadherin, was found to be up-regulated

in samples of lung-derived brain metastases, suggesting a role for E-cadherin modulation in the process of cerebral

dissemination (17) Commonly, overexpression of the HER2

gene in breast cancer is shown to be associated with TGFβ signaling, leading to the activation of Snail, Slug and ZEB1 (18) Suppression of this signaling by cucurbitacin B in mouse models led to reversal of the EMT process and reduction of brain metastases (18) Moreover, a link between TWIST and other regulators of EMT such as Snail, Slug and ZEB has been seen (16) Induction of ZEB1 by TGFβ was dependent on cooperation between Snail and TWIST in mammary epithelial cells undergoing EMT (19) TWIST, a member of the helix-loop-helix family of factors, is predominantly expressed during embryogenesis in neural crest cells It is believed that TWIST maintains roles in suppressing expression of E-cadherin, occludins and claudin-7, and induction of pro-invasive and mesenchymal genes (16, 20-22) High TWIST expression was observed in metastatic melanoma and was considered as an independent marker of poor prognosis in these patients (23) Similar findings are reported in highly invasive ductal carcinoma, prostate cancer, esophageal squamous cell carcinoma and hepatocarcinomas (24-29) A recent study described an increased expression of EMT markers Snail and TWIST present in samples of brain metastases from lung, breast, colon and renal primary tumors (17) Similar to the TGFβ signaling pathway, downstream effectors of EGFR, namely signal transducer and activator of transcription 3 (STAT3), is linked to the activation of TWIST and subsequent promotion of EMT STAT3 is a member of a family of latent transcription factors that are activated by cytokines and growth factors (30) and is constitutively

activated in many cancers The link between STAT3 and

TWIST has been suggested in invasive breast (31),

hepatocellular (32) and gastric cancers (33) Increased STAT3

activity was also evident in models of melanoma-derived

brain metastases, which display increased activity of STAT3,

relative to primary melanoma cells (34) Furthermore, an

inhibition of STAT3 may suppress the cerebral metastases as

shown in an animal model of malignant melanoma (34)

TGFβ also elicits signaling responses via non-Smad

pathways, which are thought to complement Smad signaling

in producing an effective EMT response Activation of

non-Smad signaling pathways relies on direct interactions between

effector molecules and TGFβRI and/or TGFβRII receptors

(13) The intersection of TGFβ-mediated EMT activation and

non-Smad signaling is thought to occur under the influence of

other signaling pathways associated with Erk MAP kinases,

Rho GTPases and the PI3 Kinase/AKT pathway (13, 35, 36).

MAP kinase pathways appeared to play a significant role in

TGFβ-mediated EMT, as studies have revealed

down-regulation of E-cadherin and up-down-regulation of N-cadherin and

matrix metalloproteinase expression in response to MEK/Erk

MAP kinase activation (13, 37-41) Interestingly, the MAPK

pathway is also known to be activated by mutations in the

oncogene BRAFV600E, commonly associated with melanoma,

with a higher tendency for metastasizing to the brain (42) In

fact, a recent study demonstrated inhibition of melanoma brain

metastasis cell lines harboring a BRAFV600E mutation, using

the MAPK inhibitor vemurafenib (42) Furthermore,

down-regulation of the PI3K/Akt pathway using temsirolimus, a

mechanistic target of rapamycin (mTOR) inhibitor, reduced

proliferation of melanoma-derived brain metastases harboring

mutations in the PTEN tumor suppressor gene (42).

Additionally, TGFβ is shown to activate PI3K, which results

in a subsequent activation of the Akt kinase via integrins

(43-47) In fact, αv integrin levels in cancer cell are shown to be

positively correlated with the number of brain metastasis as

well with the rate of occurrence A recent study points to the

role of αv integrin in promoting brain metastases in cancer

cells and may be involved in early steps in the metastatic

process, such as adhesion to brain vasculature and motility.

Therefore, targeting αv integrin with intetumumab could

provide clinical benefit in treating cancer patients with brain

metastases (48) Specific genetic variations in the genes for

PI3K, PTEN, AKT and mTOR have been identified as

predictors of brain metastases in a model of NSCLC (49).

Importantly, the PI3K/Akt pathway facilitates downstream

signaling that is involved in the promotion of EMT, cell

migration and cell survival (13, 43, 50) As such, inhibitors of

this pathway have been found to hinder TGFβ-mediated

E-cadherin down-regulation, thereby halting the process of EMT

(43, 51, 52) Specifically, two multiprotein complexes of

mTOR, mTORC1 and mTORC2 have been implicated in

coordinating various cellular functions associated with EMT

(53, 54) Consistent with these findings, increased mTOR

signaling has been linked to TGFβ activity via mTORC1 and

phosphorylation of p70S6K and 4E-BP1, which subsequently result in increased protein synthesis and cell size (51) Studies

of metastatic liver (55) and colorectal cancer (56) have highlighted mTOR as an emerging target of interest in regulating tumorigenesis and metastasis With respect to cerebral metastases, components of the mTOR signaling pathway are shown to be up-regulated in models of metastatic breast cancer and suppression of these markers resulted in significantly diminished metastatic potential (57, 58) Moreover, silencing of mTOR pathway components suppresses E-cadherin expression and enhances expression of the mesenchymal marker vimentin, suggesting an important regulatory role for mTOR in the processes of both EMT and MET (57)

RhoGTPases are also known regulators of cell migration, gene regulation and cytoskeleton organization (13) TGFβ regulates Rho activity in many cell types; however, the interaction between TGFβ, RhoA, and its effector kinase Rho-associated protein kinase (ROCK), at tight junctions is most significant in the process of metastasis Signaling between TGFβ and TGFβRII results in recruitment of the E3 ubiquitin ligase Smurf1 and subsequent RhoA ubiquination and degradation at tight junctions (13, 59, 60) These observations are supported by a study utilizing a model of metastatic breast cancer, revealing a distinct role for TGFβ- mediated Par6 signaling in promoting loss of cellular polarity and morphologic transformation in mammary cells (60) Furthermore, ROCK inhibition resulted in an increased number of cells permitted to migrate through the BBB, promoting the formation of cerebral metastatic lesions (61).

Cancer Stem Cells, EMT and Cerebral Metastasis

A critical role for CSCs in cancer recurrence, maintenance and metastasis has become evident Much like normal adult stem cells, the CSC is endowed with the capacity to self- renew and differentiate (62) Furthermore, normal stem cells are active in two phases; cycling or quiescent; and, as such, CSCs may function in the same manner, potentially explaining the dormancy phase of a tumor, prior to the development of a metastatic lesion Similar to embryonic stem cells, the CSCs require a specific niche provided by the microenvironment consisting of components needed to maintain stemness and differentiation (63) CSCs may contribute to tumor metastasis by the process of EMT, in which TGFβ mediated pathways generate cells with stem- like properties (9, 64)

Aberrant signaling of the Notch, Hedgehog and catenin pathways are crucial in the maintenance and activity

Wnt/β-of CSCs, as well as the process Wnt/β-of EMT (13, 62) The critical role of Notch signaling in embryogenesis and development,

particularly in cellular patterning and allocation of cell types

to tissues strengthens the notion of its role in control of

CSCs (63) Following binding of a Notch receptor to its

specific ligands (i.e Delta or Jagged), a cleaved intracellular

domain translocates to the nucleus, activating transcription

factors and promoting transcription of downstream target

genes, such as Hes and Hey (65, 66) Signaling crosstalk

between the Notch and components of the EMT pathway

(TGFβ, Snail, Slug and ZEB) has been shown to contribute

to tumor progression (66-68) In fact, TGFβ-mediated EMT

was abrogated by knockdown of Hey1 and Jagged1 or

inactivation of Notch (69) Furthermore, Notch also induces

EMT by stabilizing Snail under hypoxic conditions, via

recruitment of hypoxia-inducible factor (HIF) 1a and

HIF-2a (70-71) In addition, elevated levels of Notch 1-3 and

Jagged1 were found to be associated with disease

progression and serve as markers for poor prognosis and

metastasis in a model of NSCLC (72) Constitutively active

Notch1 in colorectal carcinoma cell lines has been associated

with an increase in EMT and stemness-related proteins such

as Slug, Smad3, Jagged1, and CD44 (73) The role of the

Notch pathway in brain metastasis was initially described

using an animal model of breast cancer, where cerebral

metastases were associated with activation of Notch1

components, along with nuclear localization of Hey1 and

Hes1 (74) Moreover, inhibition of Notch significantly

reduces the incidence of brain metastases, particularly by

altering the CD44+/CD24–sub-population (75) Importantly,

recent evidence suggests that jagged1 and Notch signaling is

involved in establishing and promoting brain metastases

from breast cancer, in a process mediated by interleukin 1b

in astrocytes (63).

The Wnt/β-catenin signaling pathway appears to play a

crucial role in regulation of stem cells and progression of

many cancers including colon, breast and cutaneous

malignancies (76-79) It is worth mentioning that Wnt also

plays an important role in normal brain development,

therefore activity of this pathway in metastases may suggest

emulation, providing further evidence for the “seed and soil”

hypothesis, as suggested by Fidler (80) The clinical

significance of the canonical β-catenin-dependent pathway

pertains to its involvement in cellular proliferation,

differentiation and survival as well as in stem cell

maintenance and reprogramming (81, 82) In addition, the

Wnt signaling pathway regulates TGFβ-mediated EMT

through its interaction with E-cadherin repressors including

Snail, TWIST and ZEB (83, 84) E-cadherin normally exists

in a complex with β-catenin at the cell membrane Therefore,

loss of E-cadherin during EMT allows β-catenin to

translocate to the nucleus in order to stimulate transcription

of regulatory genes involved in cellular proliferation and

differentiation (82, 85) Further crosstalk between the Wnt

and TGFβ signaling pathways is evidenced by the close

interaction of Smad and transcription factors induced by the Wnt pathway (86, 87) In a model of lung cancer, increased levels of dishevelled-3 mRNA in pleural effusions, suggesting this as a possible marker for micrometastases (88) Expression of dishevelled-1 and dishevelled-3 has been found to be increased in lung-derived brain metastases as well (89) A recent study of triple-negative breast cancer provided evidence of up-regulated Wnt pathway activity, identifying this as a marker of poor prognosis and metastatic disease, particularly to the lung and brain (90) Furthermore, up-regulation of Wnt/β-catenin activity in brain metastases from basal-type breast carcinoma have been documented, as evidenced by gene expression analysis (91) On the other hand, down-regulation of Wnt/β-catenin signaling in the luminal B subtype of breast cancer prevents metastasis to the brain, strengthening its role in this process (91) Therefore, targeting this pathway may prove effective in inhibiting the development of brain metastases In fact, monoclonal antibodies against Wnt ligands and associated receptors are currently being tested for their ability to inhibit tumor growth, though most trials are still in early stages (32) The sonic hedgehog (SHH) pathway is known to be a critical regulator of embryogenesis, body patterning and cancer progression (92) In the presence of SHH, smoothened proteins are released and phosphorylated to promote the activation of glioma-associated oncogene homologs (GLIs), which subsequently regulate the expression of a multitude of target genes (93, 94) The interaction of SHH, Wnt and TGFβ pathways is involved in regulation of the process of EMT For example, in fibroblasts and keratinocytes, up-regulation of TGFβ signaling revealed Smad3-dependent activation of GLI1 and GLI2 (95) Activated GLI2 was associated with loss of E-cadherin and the potential to form bone metastases

in a melanoma model (96) In vitro studies of the SHH

pathway and EMT have revealed GLI1-mediated suppression

of E-cadherin expression via induction of Snail (97) Studies

have shown the SHH/GLI pathway to be a critical regulator

of EMT, facilitating recurrence and metastasis, as well as chemotherapy resistance in models of squamous cell lung carcinoma and NSCLC (98-100) While there is limited evidence for the involvement of SHH in promoting brain metastases, a study conducted using samples of six metastatic brain tumors demonstrated increased expression of downstream mediators of the SHH pathway, particularly GLI1, which correlated positively with expression of Snail, and negatively with expression of E-cadherin, suggesting a role for EMT in brain metastases (101)

Role of Micro RNA in Cerebral Metastasis

miRNAs are small, non-coding, single-stranded RNAs that regulate gene expression by targeting mRNA transcripts, leading to their translational repression or degradation (102).

A single miRNA can simultaneously regulate multiple genes,

resulting in complex functional outcomes (103) The

identification of groups of genes targeted by the same

miRNA provides insight into the cross-talk between multiple

signaling networks and their role in controlling diverse

biological processes Numerous miRNAs are now being

identified for their role as oncogenes or tumor suppressors

(104) Furthermore, extensive research has provided

evidence that several microRNA are involved in the process

of EMT; many of these having a role in mediating cell-cell

adhesion, cytoskeletal arrangement or oncogene expression

(102, 103, 105) Recently, much of the work regarding

miRNA in EMT regulation focuses on the miR-200 family,

which includes miR-200a, miR-200b, miR-220c, miR-141,

and miR-429 (105) This association was realized upon the

finding that the cells undergoing EMT in response to TGFβ

had noticeably reduced miR-200 expression (15).

Specifically, members of the miR-200 family were found to

exert a negative regulation on ZEB1 and SIP1 expression,

suggesting that down-regulation of these miRNA is required

for initiation of EMT (15) This observation was further

supported by the fact that members of the miR-200 family

repress EMT by silencing ZEB1 and ZEB2 in gastric and

breast cancer (106-108) In addition, miR-429 expression has

been associated with down-regulation of mesenchymal

markers including MMP2, Snail and ZEB2 (109).

Interestingly, induced expression of miR-200 correlated with

increased levels of E-cadherin mRNA, indicative of the

reversal process of EMT, MET (15) In metastatic NSCLC

cells, the expression of miR-200 correlated with reduced

gene expression, particularly related to genes involved in cell

signaling, invasion and proliferation (110)

Recently, miRNA expression has become a topic of interest

as it pertains to mediating pathways involved in

tumorgenicity as well as CSC differentiation, self-renewal

and maintenance (111) Of interest, 107, miR153,

miR-204 and miR-218 are shown to influence the self-renewal and

maintenance of glioma stem-like cells (112-115) In a model

of breast cancer, Lin28-mediated repression of let-7 was

associated with CSC production (116) Expression of miR-7

in metastatic breast CSCs was correlated with increased

expression of the pluripotency gene KLF4, suggesting an

important role for miRNA in CSC stemness and metastasis

(117) In concordance with these studies, a pivotal role for

microRNA in CSC maintenance has also been reported in

models of colorectal, lung and hepatocellular carcinoma

(118-121) Moreover, a recent review provided evidence for a 30

miRNA signature correlating with expression in the TGFβ,

Notch and Wnt signaling pathways, implicating their role in

regulating EMT and CSCs (103)

Recently, groups of miRNA have been identified as unique

markers for metastatic tumors, suggesting a role for miRNA

in developing organ-specific metastases Importantly, a recent

study using expression-based profiling of miRNA was able to accurately identify the primary tumor of origin of brain metastases in 84% of samples, suggesting an important role

in diagnosis (122) Also, a group of miRNAs were described that are exclusively expressed in metastatic tumors based on the analysis of 336 cancer samples from 22 unique sites, suggesting a role in site specific metastasis (123)

While the precise mechanism by which the cerebral microenvironment interacts with tumor cells has yet to be understood, studies suggest a possible interaction between the astrocytic milieu and tumor cells, in which complex alterations of miRNA expression could take place (124) In support of this theory, a recent study demonstrated the ability

of astrocytes to alter the microRNA expression patterns of lung cancer cells when co-cultured together (125) Specifically, co-cultured cells exhibited reduced expression

of miR-768-3p, which was linked to increased cell viability

via increases in K-ras (125) Additionally, a study of breast

and bone metastatic models revealed down-regulated miR-7 expression in CSC derived from metastatic tumors, as well

as an inverse relationship between miR-7 and the pluripotency gene KLF4 (117) Interestingly, this inverse relationship between miR-7 and KLF4 was also associated with metastasis-free survival only in brain metastases, demonstrating a site-specific interaction between microRNA, CSCs and the cerebral microenvironment, leading to prognostic implications Global patterns of gene expression demonstrated an up-regulation of hsa-miR-17-5p in triple- negative breast cancer tissues in The Cancer Genome Atlas (TCGA) In addition, a negative correlation between hsa- miR-17-5p and overall survival as well as PTEN and BCL2 target genes was observed in TCGA breast cancer specimens (126) Other miRNAs were found to have roles in tumor cell invasion and extravasation through the BBB Specifically, miR-1258 was shown to regulate expression of heparanase,

a pro-metastatic enzyme stored in endothelial and glial cells, involved in breakdown of heparan-sulfate chains, rendering cells more capable of crossing the BBB (127, 128) In addition, miR-22 and miR-378 mediate expression of MMP-

2, MMP-9 and VEGF, implicating a cross-talk between the tumor cells, extracellular matrix and vasculature in facilitating invasion and establishing secondary lesions in the brain Interestingly, aberrant expression of miR-10b, miR- 29c, miR-145, miR-146a, miR-200, miR-210, miR-199a/b and miR-768-3p were discovered in cerebral metastatic lesions from multiple primary tumors, suggesting the pivotal role of miRNA in brain metastasis, which has potential to aid

in diagnosis, prognosis and discovery of therapeutic targets Involvement of approximately 38 distinct miRNA associated with cerebral metastases from different primary tumors (NSCLC, breast, CRC, melanoma and renal tumors) are presented in Table I (129-165) Of these miRNA, 25 were found to have increased expression, 10 were found to

have decreased expression, and 3 were found to have

variable expression, when compared to their matched

primary tumor counterparts (Table I).

Genetics Associated with Cerebral Metastasis

Studies of gene expression have suggested the existence of

a genetic signature present in primary tumors that defines

their metastatic potential (166) While, much debate has

since emerged regarding the significance of genetic

signatures and their role in metastasis, Fidler and colleagues

suggested that the process of metastasis requires cancer cells

to acquire additional mutations in order to develop metastatic

potential (167) Furthermore, it has been demonstrated that

while a genetic signature for poor prognosis may exist, an

additional set of genetic aberrations contributes to the

site-specific dissemination of metastatic tumor cells (168) Rather

than directing the process of metastasis, this study suggests

that the genes corresponding to poor prognosis may instead

provide tumor cells with baseline metastatic properties with

a phenotype encouraging metastasis (168) Alternatively, a

model of breast-derived brain metastases suggests that it is

not a genetic signature that predetermines a tumors clinical

course, but rather a unique interaction between the tumor and

its microenvironment that contributes to disease progression

(80) Given the significance of genetic markers in defining

tumor progression and metastasis, it is prudent to detail

genetic markers as they pertain to cerebral dissemination

from the breast, lung, skin, colon and kidney (See Figure 1

for details).

Breast

Approximately 10-30% of breast cancer patients will develop

cerebral metastases (169) In patients with metastatic breast

cancer brain metastases were shown to have the worst

prognosis (7.35 months), followed by metastases to the liver

(36.7 months), bone (44.4 months) and lung (58.5 months).

Brain metastases from breast cancer can be stratified based

on hormone receptor status About 25% of patients with

breast cancer have an amplification in HER2 and, of these,

30-55% develop metastatic brain lesions (170-174) This risk

is elevated in the setting of hormone-receptor negativity

(175, 176) Median survival of patients with HER2-positive,

ER-negative brain metastases has been found to be

approximately 28 months (177) A study of 66 patients with

HER2 breast cancer displayed good performance status,

controlled extracranial disease and single brain metastases

had better outcome (178) Patients with triple-negative breast

cancer (ER–/PR–/HER2–), on the other hand, are at increased

risk of first recurrence of cerebral metastasis (179) with a

tendency to cluster early in the patient’s disease trajectory

(175) These patients are at a 25-46% risk of developing

CNS metastasis (180-182) with a survival time of less than six months (179, 182, 183).

In a model of breast cancer, gene expression analysis revealed 243 genes that were differentially expressed in metastatic cell lines, of those, 17 genes were highly correlated with brain metastasis (184) More importantly, these genes did not coincide with those involved in metastasis to other

organs Among the 17 genes, COX2, EGFR ligand HBEGF

and the a2,6-sialyltransferase ST6GALNAC5 were identified

as mediators of homing, cancer cell migration and passage through the BBB Findings also indicate that in breast cancer,

a long period of remission often precedes distant relapse, supporting the notion that breast cancer cells initially lack the full competence for outgrowth in distant organs but develop this under the selective pressure of different organ microenvironments (168, 184-186) Alternatively, a recent study of 18 primary and 42 breast cancer-derived brain

metastases found mutations in the TP53, PIK3CA, KIT,

MLH1 and RB1 genes, within which no mutations were found

to be unique to cerebral metastases (187) Interestingly, in a matched pair of primary tumor and metastatic brain lesion, a mutation in p53 was discovered, however the acquisition of additional mutations might have occurred during the metastatic process (187) Mutations in TP53 were also found with a higher frequency in metastatic tumors, compared to primary breast cancer cells (187, 188) A similar study, using expression profiling of 23 matched sets of brain metastases and primary breast tumors, found DNA double-strand break

repair genes BARD1 and RAD51 to be up-regulated in

samples of metastatic lesions, suggesting a role for these genes in evading the effects of reactive oxygen species in the brain (189) Studies of epigenetic gene regulation have revealed differential methylation patterns of genes exist between brain metastases and their primary tumor counterparts (190, 191) Importantly, one study found the

BNC1 gene to be more frequently methylated in metastatic

tumors than in primary breast cancer (190) BNC1 is a target

of TGFβ in mediating EMT, as its expression is preserved in primary breast tumors while silent in metastatic lesions (191) This suggests its initial role in promotion of EMT, thereafter possibly contributing to MET while establishing secondary lesions (190)

Lung

The pathogenesis of lung cancer exists in two broad clinical subtypes, namely non-small cell carcinoma (NSCLC), representing approximately 75-85% of tumors, and small cell lung carcinoma (SCLC) accounting for the remaining 15-25% Of these, approximately 30-50% are likely to develop cerebral metastases; where 25% arise from NSCLC (192, 193) The development of cerebral metastases is considered an indicator of advanced disease and poor

prognosis Without treatment, median survival time ranges

from only 1-2 months, expanding to 4-6 months with the

addition of radiotherapy (193-195) Improved survival in

patients with single brain metastases derived from NSCLC

has been shown with the addition of neurosurgical resection

and/or radiosurgery with mean survival times approaching

12-14 months (193)

A study comparing expression data between 16 metastatic brain tumors with 37 primary NSCLC samples revealed 244 genes with altered expression levels, corresponding to genes involved in adhesion, cell-cell communication and motility (196) A similar study, comparing brain metastases from NSCLC to non-metastatic lung tissue assessed 17,000 genes and found 1,561 to have altered expression, showing genes involved

Table I microRNAs involvement in brain metastasis

miRNA Primary site Pattern of Proposed role in metastasis Reference expression

miR-1 CRC h Interaction with MACC1 129, 130

miR-7 BC i Increased KLF4 expression 117

miR-9 BC; CRC h Inhibition of E-cadherin 131, 132 miR-10b BC; CRC; ccRCC h BC Modulation of HOXD10, TIAM1, MICB, TIP30, 129, 133, 134 h CRC Twist and E-cadherin expression

i ccRCC miR-15b Melanoma h 135

miR-16 Melanoma h Inhibition of EMT via phosphorylation of FAK and Akt proteins 135, 136 miR-19a BC i Regulation of cyclin D1, Bim, TNFa and PTEN expression 137, 138 miR-20b BC h Suppression of PTEN 139, 140 miR-21 NSCLC h Downstream mediator of STAT3 141

miR-22 CRC h Modulation of TIAM1, MMP-2, MMP-9 and VEGF expression 129, 142 miR-28 CRC h Targeting of CCND1 and HOXB3 expression 129, 143 miR-29c BC; i BC Epigenetic regulation of tumor-related genes; 137, 144, 145 Melanoma i Melanoma Targeting of TIMP3, PDCD4 and RASA1 miR-31 CRC i Downstream effector of TGFb that targets TIAM1 129, 146 mIR-95 NSCLC i Suppression of cyclin D1 147

miR-125b CRC h Suppression of LIN28B 129

miR-126 CRC h Inhibition of the RhoA/ROCK signaling 129, 148 miR-133a/b CRC h Downstream target of TPp63 with inhibitory effect on RhoA, 129, 149 E-cadherin and vimentin miR-143 CRC h Regulation of MACC1 expression 121, 129 miR-145 NSCLC; CRC h NSCLC Targeting of OCT4, EGFR, c-myc, MUC1, TPD52 and NUDT1 129, 150, 151 i CRC miR-146a BC; CRC i BC Modulation of b-catenin and hnRNPC expression 129, 152 h CRC miR-150 Melanoma h Targeting of c-Myb 135, 153 miR-184 NSCLC h Inhibitor of c-myc and CCND1; induction of p15 and p21 154, 155 miR-197 NSCLC h Negative regulator of FUS1 154, 156 miR-199a/b CRC; ccRCC h CRC Regulation of HES1; inhibition of c-Met 129, 134, h ccRCC 157, 158 miR-200 BC; Lung h BC Targeting of ZEB1 and ZEB2 159

h Lung miR-210 BC; Melanoma h BC Targeting of MNT; Induction of angiogenesis after hypoxia 137, 160, 161 h Melanoma miR-328 NSCLC h Up-regulation of PRKCA 162

miR-374 Melanoma h 135

miR-378 NSCLC h Up-regulation of MMP-2, MMP-9 and VEGF 163

miR-509 BC i Suppression of RhoC and TNFa 164

miR-542 Melanoma i Down-regulation of PIM1 165

miR-576 CRC h 129

miR-768-3p BC; Lung i BC Targeting K-ras 125

i Lung miR-1258 BC i Inhibition of heparanase 127

HS_170 CRC i 129

HS_287 CRC h 129

in adhesion, motility and angiogenesis to be up-regulated, and

genes involved in apoptosis and neuroprotection to be

down-regulated (197) Furthermore, analysis of 12 candidate genes

from samples of NSCLC, assessing the correlation between

gene expression and occurrence of brain metastases found three

genes, CDH2, KIFL1 and FALZ, to be predictive of brain

metastases (4) Importantly, CDH2 is known to regulate

adhesion, and is a mediator of EMT, suggesting its role in this

process (4, 198, 199) Similarly, loss of LKB1 and mutation of

KRAS have been shown to be predictive of brain metastases in

NSCLC (200) Recent genomic analysis using comparative

genome hybridization techniques has revealed numerous copy

number variations (CNV) predictive of brain metastases from

primary NSCLC (201, 202) An increase in the number of

CNVs has been observed in secondary tumors in comparison to

their primary counterpart, suggesting a degree of genetic

variability that takes place during the metastatic process (201)

A recent study has demonstrated that about 21.9% patients

with NSCLC showed the expression of programmed cell

death-ligand 1 (PD-L1) in brain metastases Furthermore,

PD-L1 positivity in brain metastasis samples was seen in

patients with heavy smoking history as well as

radio-therapeutic treatments giver prior to surgery (203)

Interestingly, a newly discovered rearrangement of

anaplastic lymphoma kinase (ALK) is seen in about 2-7% of

NSCLC (204) and has recently become a marker of interest for targeted chemotherapy The propensity for ALK-rearranged NSCLC to metastasize to the brain has been a subject of contention, with several studies citing greater likelihood (205), however others show no significant increase in brain metastases (206) Crizotinib, a first-generation ALK inhibitor,

is shown to be highly efficacious against ALK-positive NSCLC and is currently approved for first-line treatment (207, 208), however it is important to consider that CNS progression develops in up to 60% of patients treated with this chemotherapeutic agent (204) The pattern of CNS progression

in patients receiving crizotinib was studied by Costa et al., who

discovered poor CSF concentrations of the drug, due, in part,

to both passive diffusion restriction and active efflux via

P-glycoprotein (209, 210) Several second generation inhibitors, such as alectinib, ceritinib, and brigatinib, are currently in clinical trials, with positive results due to better CNS penetrance, however, ongoing preclinical studies are awaiting outcome (211-213) Perhaps the novel of ALK- inhibitors is the third-generation, loratinib, believed to be effective against all resistant mutants of ALK+ NSCLC (214, 215) Clinical trials for this novel chemotherapeutic agent are currently underway, with published evidence from one patient indicating a favorable treatment response and re-sensitization

ALK-to crizotinib (211, 214)

Figure 1 Genes associated with cerebral metastasis from distinct organ sites Figure represents the alterations in unambiguous genes linked with

brain metastasis from breast, lung, kidney, colon or skin